Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1’s mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation—contrasting siRNA’s delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner. Key features • The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics. • Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells. • AID’s application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment. • The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.

This protocol is used in: J Cell Biol (2023), DOI: 10.1083/jcb.202208018 The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation.Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells.The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein.The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation.We have used AID to explore the mitotic functions of the replication licensing protein CDT1.Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment.Using live imaging, we found that mitosisspecific degradation of CDT1 delayed progression and chromosome mis-segregation.AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1's mitotic roles.The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation-contrasting siRNA's delayed RNA degradation and protein turnover.In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts.In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner.

Background
Conditional protein degradation is an invaluable approach to understand cellular function.Among the various techniques available, the auxin-inducible degron (AID) system stands out.AID is a versatile molecular tool extensively utilized in cell biology and molecular genetics for the conditional destabilization of target proteins, facilitated by CRISPR/Cas9 gene knock-in technology [1,2].This mechanism capitalizes on the unique ability of the plant hormone auxin to rapidly degrade specific proteins bearing an AID sequence not only in non-plant cells like DLD1, HCT116, and HeLa but also in organisms like Caenorhabditis elegans, mouse, and yeast [3][4][5][6].The AID assay furnishes a valuable means for probing protein functions and regulating protein expression in live cells.Its precise control over protein degradation addresses limitations of other methods such as RNAi gene silencing, which is both slow and less specific, lacking conditionality.The AID system, on the other hand, operates at the protein level, offering simplicity, rapidity, effectiveness, and reversibility.Central to this system is the interaction between the transport inhibitor response 1 receptor (TIR1), an F-box related protein, and the AID tag (available as 25 or 7 kDa), genetically fused to the protein of interest [7][8][9].The half-life degradation of the tag, which is approximately 30 min, can be observed in a mammalian cell line expressing TIR1.Upon auxin binding to the TIR1  Software and datasets Open the full-length gene, including both introns and exons, and locate the translation start (ATG) and stop site (TGA) based on the position in the RefSeq (which is NCBI database).3.For N-terminal tagging, select and copy approximately 15 base pairs upstream of the ATG and approximately 150 base pairs downstream from the ATG.This sequence window will provide options to choose the best gRNA near to the point of modification.4. For C-terminal tagging, select and copy approximately 15 base pairs downstream of the TGA (stop codon) and approximately 150 base pairs upstream from the stop codon as stated earlier. 5. Paste the selected sequence into the Crispor or/and CHOPCHOP sgRNA design tool, provided by Broad institute.Do not forget to select appropriate organism genome, which provides SG score, off-target, and valuable parameters.6. Download the results of the sgRNA design as a .txtfile and import it into a spreadsheet.7. Review the sgRNA results obtained from the design tool and prioritize guides that cut within 50 base pairs after the ATG (for N-terminal modification) or close to Stop codon (for C-terminal modification), giving preference to those with none or fewer off-targets.8. Focus on specific columns, such as "Position of Base After Cut (1-based)," "sgRNA Target Sequence," "On-Target Rank," and "Off-Target Rank" to select guides with better scores.9. Pick at least three high-ranking guides for the cloning into SG backbone plasmid (Table 1).

C. sgRNA annealing and ligation
1. Prepare stock solutions of the gRNA primers.
a.The gRNA primer contains 120 µg of DNA and was diluted in 200 µL of Milli-Q water to achieve a concentration of 0.6 µg/µL.Dilute gRNA primer 1:100 to achieve a concentration of 6 ng/µL.b.The reverse gRNA primer contains 170 µg of DNA and was similarly diluted in 200 µL of Milli-Q water to achieve a concentration of 0.85 µg/µL.Dilute reverse gRNA primer 1:100 to achieve a concentration of 8.5 ng/µL.2. Prepare the annealing reaction of each pair of oligos in annealing buffer as indicated in Table 4 and Table 5.

E. Screening of the clones
1. Examine plate for colony formation.2. On a new LB + Amp plate, use a marker to draw a 4 × 5 grid on the backside of the plate.Streak single colonies into their own spot on the grid.3. Incubate grid plate in a 37 °C incubator overnight.4. Next day, inoculate 4-5 colonies that grew on the grid into 10 mL of LB + Amp broth in 50 mL Falcon tubes to isolate the plasmid for sequencing. 5. Place Falcon tubes in a 37 °C shaking incubator at 200 rpm overnight.6.Next day, isolate the plasmids using Plasmid Isolation kit (Qiagen) following manufacturer's protocol.7. Send plasmid samples (Table 8) for sequencing to confirm the clone.

F. Synthesizing Cdt1 gene and the AID+YFP tag into the repair template vector
We synthesized a DNA fragment containing 800 bp (between 500 and 1 kb) homology regions on each side of Cas9 cut site in the Cdt1 sequence; AID with YFP sequence was inserted in between these two homology arms (synthesized by Gene Universal).We then cloned this product into the vector HJURP using KpnI-HindIII restriction sites (a kind gift from Dr. Daniel Foltz, Northwestern University, Evanston, IL, USA).The PAM

Validation of protocol
This protocol or parts of it has been used and validated in the following research article: Rahi et al. ( 2023).The Ndc80-Cdt1-Ska1 complex is a central processive kinetochore-microtubule coupling unit.J Cell Biol.(Figure 1, panel B, Figure S1, panel B) [12].

General notes
1. Make sure the PAM sequence is mutated in the donor plasmid/repair template.
2. Make sure that the TIR1+ mammalian cells (DLD1/HCT-116/HeLa/293T) being used are appropriate for the phenotypic or functional characterization experiments to be adopted.

Troubleshooting
Problem 1: Selection between heterozygous and homozygous clones.Solution: For homozygous selection (biallelic), use two donor plasmids containing different selections (e.g., one plasmid with GFP and the other with m-Cherry).Cells can be sorted after insertion to confirm they contain two colors.
Problem 2: Difficulty inducing protein degradation by auxins.Solution: Optimize concentration of auxins, starting with the lowest concentration and then increasing concentrations.
Problem 3: Auto/leaky degradation without the addition of auxins.Solution: Use mini-AID (7 kD) or mutant form of AID (AID2) that is still able to properly bind to auxins (IAA/5-Ph-IAA).

b. Mix 1
µL of ladder with 1 μL of 6× loading dye and 4 µL of Milli-Q water.Load the entire volume (50 µL) on gel.Mix 2 µL of uncut pX330 plasmid sample with 0.4 µL of 6× loading dye.Load the entire volume on gel.c.Mix 2.5 µL of BsbI cut pX330 plasmid sample with 0.5 µL of 6× loading dye.Load the entire volume on gel.d.Run gel at 90 V for 45 min.

Published: Jan 20, 2024 c
. Allow mixture to cool before adding 5 µL of ethidium bromide for a 0.5 µg/mL concentration from 10 mg/mL stock.Swirl flask to mix.d.Pour solution into casting gel tray and insert well comb.Allow gel to solidify at room temperature (RT).

Cloning of SG sequence at Bbs1 site into pX330 vector to generate sgRNA constructs
Select sgRNA sequences based on the target region: order oligos for sense and antisense strands, including the sgRNA target sequence and its reverse complement (Table2 and Table 3).
Note: The sequences of SG for Cdt1 were reconfirmed using NCBI blast feature, and the first hit for all three sequences was the Cdt1 gene from Human.

Table 3 . Primer list of top three sgRNAs containing Bbs1-compatible cohesive end ready for ordering sgRNA # sgRNA sequence Forward and reverse primer sequences
Inactivate the enzyme at 65 °C for 20 min.7. Purify the digested plasmid by gel purification using the gel extraction kit.

Table 4
Prepare ligation reaction with pX330 and sgRNA as indicated in Table6.Prepare a negative control without sgRNA as indicated in Table7.

et al. (2024). Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines. Bio-protocol 14(2): e4923. DOI: 10.21769/BioProtoc.4923. 9 Published: Jan 20, 2024Table 7 . Negative control
μL of the ligated plasmid or the vector control to 50 μL of Stbl3 cells.4. Incubate bacterial and DNA mixture on ice for 20 min.5. Heat shock in a 42 °C water bath for 45 s. 6. Cool mixture on ice for 2 min.7. Add 400 μL of S.O.C. medium to mixture.8. Place in a 37 °C shaking incubator at 200 rpm for 1 h.9. Spin tube at 13,000 rpm for 1 min at RT to pellet.10.Remove 300 μL of media from the pellet.Resuspend pellet in the remaining volume of media.11.Plate 100 μL of cells on LB + Amp plate (Ampicillin working concentration 100 μg/mL) using sterilized spreader.12. Label plate and incubate in a 37 °C incubator overnight.